Reduced amyloid A degrading activity (ADA) in human serum has been found to correlate positively with a diagnosis of secondary amyloidosis. Ravid M., Greenman Y., Shapira J., Kedar I., Israel Journal of Medical Sciences, 26, 191-194 (1990); Wegelius O. Teppo Anna-M, Maury CPJ, British Medical Journal, 284, 617-619 (1982); Maury CP, Teppo Anna-M, The Lancet, July, 234-237 (1982).
Amyloid A is one member of a heterogenous group of proteins known generally as amyloids which share certain characteristics, including the binding of sodium diphenyldiazo-bis-alpha-naphthylamine sulfonate (Congo red) and the consequent exhibition of green birefringence under polarized light; a .beta.-pleated sheet structure as revealed by x-ray diffraction; and a fibrillar appearance with periodic twists when examined under the electron microscope.
Amyloidosis is a collective name for a number of diverse diseases having as a common feature the extra-cellular deposition of an amyloid protein onto affected tissues. This amyloid deposition appears to result from: 1) a reduction in amyloid degrading activity, a proteolytic activity that may be attributable to one or more specific amyloid degrading factors (ADF) present in serum and certain tissues and/or 2) increased synthesis of amyloid precursor proteins. Kedar I, Sohar E, Ravid M, Journal of Laboratory and Clinical Medicine, 99:693-700 (1982); Teppo A-M, Maury CPJ, Wegelius O. Scand.J.Immunol., 16, 309-314 (1982); Shirahama T., Muira K., Ju S-T, Kisilevsky R., Gruys E., and Cohen A. S., "Amyloid Enhancing Factor-Loaded Macrophages in Amyloid Fibril Formation." Laboratory Investigation 62:61-68, 1990.
The amyloidoses are subdivided into the following disease categories based upon clinical manifestations and the nature of the precursor protein:
(1) Primary Amyloidosis: Although not associated with known disorders, it is most likely associated with plasma cell dysfunction, as in the case of multiple myeloma. For those patients, the amyloid protein involved is derived from impaired metabolism of immunoglobulin light chains, especially from immunoglobulin .lambda. and K. PA1 (2) Secondary Amyloidosis: Within this class of amyloidosis, extra-cellular deposition of amyloid A protein in organs is often the result of chronic inflammation and/or infection accompanying diseases such as rheumatoid arthritis, familial Mediterranean fever, myeloma, and neoplastic disorders. Circulating serum amyloid A (SAA) is the precursor of AA based on amino acid sequence homology and on immunological cross-reactivity. PA1 (3) Alzheimer's Disease: Alzheimer's patients have intracerebral amyloid deposits in brain parenchyma occurring as intraneuronal neurofibrillary tangles, extracellular plaques and cerebrovascular amyloid deposits. The deposit is a 4KD protein known as .beta.A4 or A.beta.P (hereinafter A.beta.P) which derives from an amyloid precursor protein. PA1 (4) Endocrine-related Amyloidosis: Amyloid deposits are localized in the endocrine organs and/or specific cell groups of the endocrine gland. The amyloid protein deposited in these organs and/or cells often includes a polypeptide sequence homologous or identical to a portion of the polypeptide chain from related hormones, such as amylin found in the islets of Langerhan in Type II diabetes, or procalcitonin found in medullary carcinoma of the thyroid. PA1 (5) Other Amyloidotic Diseases: (a) Systemic Amyloidosis results from regular, long-term hemodialysis treatment. The amino acid structure of the amyloid deposit in these patients is homologous to that of .beta..sub.2 -microglobulin; and (b) Senile-Related Amyloidosis, an amyloidotic condition in which an amyloid protein originating from prealbumin is deposited in the heart, aorta and other vessels. Patients suffering from familial polyneuropathy are also affected by an amyloid protein derived from prealbumin. PA1 (1) The measurement of the cleared area on agar can be subjective, compromising the reliability of the assay. In contrast, spectrophotometric ADA measurement yields objective, precise results; PA1 (2) The existing method requires an amyloid A sample size of at least from two to twelve times that required by the method of the invention. This is significant since amyloid A is not readily available commercially and is both costly and time consuming to prepare. Further, the gel diffusion methods result in the loss of unreacted amyloid A in the gel. In the method of the invention, unreacted amyloid A can be easily recovered for use in future assays; PA1 (3) With the existing method, ADA depends upon diffusion of the serum through the amyloid A containing agar. If, as is thought, ADA is an enzymatic reaction, factors affecting enzymatic reactions such as substrate concentration, product inhibition, pH, ion concentration, etc. are difficult to control in a gel medium. Furthermore, incorporation of the amyloid A evenly into the agarose solution can only be achieved over a narrow temperature range, since agarose solution gels readily upon cooling while amyloid A protein denatures if overheated. With the method of the present invention, on the other hand, the ADA reaction proceeds in an aqueous medium where chemical and physical conditions are favorable for enzymatic reactions and relatively easy to control; PA1 (4) With the existing method, the gel diffusion process takes 16 to 24 hours. In contrast, the ADA assay of the present invention requires only 20 to 30 minutes of incubation time; and PA1 (5) The method of the invention can be adapted for use in conjunction with other available assays for prognosis of secondary amyloidosis. The method of the present invention can also be used as a research tool for studying amyloid A metabolism and exploring potential therapies for secondary amyloidosis. On the other hand, the existing gel diffusion assay for ADA is difficult to combine with other available assays, and it is impractical to analyze the reaction products, impairing the utility of the existing ADA assay for research purposes.
Secondary amyloidosis is known to affect many animals, including human and non-human primates, dogs, cats, bovines, golden hamsters, birds, snakes and honeybees. At present, accurate clinical diagnosis of secondary amyloidosis requires biopsy of abdominal fat, rectal tissue, or renal tissue, wherein the excised tissue is examined for amyloid A deposits. The tissue biopsy involves a painful surgical operation and carries the risks inherent to any invasive procedure. As a result, diagnosis of secondary amyloidosis in human patients frequently occurs at the later stages of the disease when organ enlargement is palpable and when the involvement of affected organs is extensive. At that stage of the disease, amyloid A infiltration is often irreversible, and death due to organ dysfunction can result.
ADA has been measured with agarose gel diffusion methods such as those described by Kedar I, Sohar E, Ravid M, Journal of Laboratory and Clinical Medicine, 99, 693-700 (1982) and Maury CPJ, Teppo A-M, Salaspuro MP, Clinical Chimica Acta, 131, 29-37 (1983). Briefly, the methods of Sohar and Maury involved first isolating and purifying a quantity of amyloid A protein. The purified amyloid A is incorporated into an agarose solution which is then poured into a Petri dish or onto a glass plate to form an opaque gel. Sample serum is allowed to diffuse into the gel through wells, followed by a room temperature incubation for 16 to 24 hours. ADA is indicated by the development of a clear, transparent zone adjacent to the wells. The approximate level of ADA is estimated by measuring the diameter of the cleared area.